JPH08154671A - Hybridoma 28k29 - Google Patents

Abstract

PURPOSE: To obtain the subject hybridoma capable of producing a human-type monoclonal antibody useful for the diagnosis and remedy of lung cancer. CONSTITUTION: This Hybridoma 28K29(FERM-P-14119) is capable of producing a human-type monoclonal antibody specifically reactive with lung cancer. This hybrldoma is obtained by cell fusion of myeloma cells with in vitro stimulated human lymphocytes. The produced human monoclonal antibody can react with the cell membrane antigen of a lung cancer cell strain, react with lung cancer tissue in immune tissue staining, react with the humoral component of lung cancer patients, have complement-dependent cytotoxlc activity and bring about positive reaction and the degeneracy of cancer site in an enrichment experiment to cancer site with a cancer-carrying mouse.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a human type monoclonal antibody which specifically reacts with lung cancer and reacts with a cell membrane antigen of a lung cancer cell line.
Contract number FERM P at Institute of Biotechnology, Institute of Industrial Technology
Hybridoma 28K29 having the characteristics of the cells deposited under No. 14119, the humanized monoclonal antibody produced by the hybridoma 28K29 having the characteristics of the cells deposited under the deposit number FERM P-14119 at the Institute of Biotechnology, Institute of Industrial Science, The present invention relates to a cancer diagnostic agent containing the human type monoclonal antibody or the human type monoclonal antibody fragment as a main component, and a cancer therapeutic agent containing the human type monoclonal antibody or the human type monoclonal antibody fragment as a component.

[0002]

2. Description of the Related Art Immunological diagnosis, treatment and prevention methods using antibodies are highly desired clinically, and in particular, have clinical value in disease areas such as autoimmune diseases, cancer and infectious diseases. high.
According to the vital statistics data of the Ministry of Health and Welfare, the number one cause of death in Japan is cancer, and the number of deaths due to cancer is increasing year by year. Among them, the number of deaths due to lung cancer / colorectal cancer is increasing remarkably, and development of cancer diagnostic agents / cancer therapeutic agents against them is strongly desired in clinical practice.

As an immunotherapeutic agent for cancer, a mouse type monoclonal antibody (Dippold, WG et al., P
rc. Natl. Acad. Sci. USA. , 7
7, 6114-6118, 1980; Korowsk.
i, H et al., Proc. Natl. Acad. Sci. U
SA. , 81, 216-219, 1984; Hells.
trom, I et al., Proc. Natl. Acad. Sc
i. USA. , 82, 1499-1502, 1985).
Alternatively, a human-mouse chimeric antibody (Neuberge
r, M. S et al., Nature, 312, 604-60
8, 1984; Morrison, S .; L, Scien
CE, 229, 1202-1207, 1985) and the like.

However, an in-vivo diagnostic agent or therapeutic agent containing a heterologous antibody such as a mouse monoclonal antibody or a human-mouse chimeric antibody or the antibody fragment is foreign to humans and may cause side effects such as anaphylactic shock. High (Mitsuyo Honda et al., Bio
therapy, 4, 844-847, 1990), clinical utility is very low.

On the other hand, if human type monoclonal antibodies are used, the safety for humans is markedly improved, and it is an ideal drug as an in-vivo diagnostic drug or therapeutic drug.
However, techniques for producing human-type monoclonal antibodies are extremely difficult, and various attempts have been made to stably and in large amounts obtain human-type monoclonal antibodies that react with cancer (Irie, RF, et al., Proc. Natl.A
cad. Sci. USA. , 79, 5666-567
0,1982; Yamaguchi, H et al., Proc.
Natl. Acad. Sci. USA. , 84, 241
6-2420, 1987; Richard J. et al. Cot
e et al., Proc. Natl. Acad. Sci. US
A, 80, 2026-2030, 1983; Susan.
-P. C. Cole et al., Cancer Res. Four
4, 2750-2753, 1984; Martin.
V. Haspel et al., Cancer Res. , 45,
3951-3961, 1985; Richard J. et al.
Cote et al., Proc. Natl. Acad. Sci.
USA, 83, 2959-2963, 1986; Llo.
yd.H. Smith et al., Human / Hybrido
ma (Anthony J. Strelkauskas
Edited, p121-158, 1987, Marcel De
kker Inc. , New York); Karol
・ Sikora, Human ・ Hybridoma (A
nthony J. Edited by Strelkauskas, p1
59-181, 1987, Marcel-Dekker.
-Inc. , New ・ York); Anthony ・
J. Strelkauskas et al., Human / Hyb
ridoma (Anthony J. Strelkau
edited by skas, p227-251, 1987, Marce.
1-Dekker-Inc. , New York); T
homes.B. Kjeldsen and others, Cancer
Res. , 48, 3208-3214, 1988), and there is no clinically practical example.

Further, among cancers, the number of deaths due to lung cancer is rapidly increasing and the 5-year survival rate after surgery is also very low. Therefore, a method for diagnosing and treating lung cancer is strongly desired in clinical practice. . As a cancer marker for lung cancer, sialyl Lewis X (SLX) in the case of lung adenocarcinoma,
In the case of squamous cell carcinoma of the lung, squamouse cell carcinoma antigen (squamous, cell, carcinoma,
antigen; SCC), in the case of small cell lung cancer, neuron-specific enolase (neuron specificity)
c. enolase; NSE), and carcinoembryonic ant as a whole lung cancer
igen; CEA) is often used (Hiroshi Ariyoshi, Modern Physician, 12, 981-985, 1
992) is not always satisfactory, and the antibodies used in these diagnostic agents are all mouse type monoclonal antibodies, and it is preferable to use them as in-vivo diagnostic agents or therapeutic agents in view of the risk of side effects. Absent.

[0007]

Therefore, it is an object of the present invention to develop a human type monoclonal antibody having high specificity for lung cancer and reacting with a cell membrane antigen of lung cancer cells, and to use it for diagnosis and treatment of lung cancer. Is.

[0008]

[Means for Solving the Problems] As a result of earnest research, the present inventors have found that they react with cell membrane antigens of lung cancer cell lines, react with lung cancer tissue in immunohistological staining, react with components in body fluid of lung cancer patients, and The present inventors have found a humanized monoclonal antibody which exhibits body-dependent cytotoxicity (CDC) and is positive in an accumulation test to a cancerous part in a tumor-bearing mouse and causes degeneration of the cancerous part. Completed the invention.

That is, the human type monoclonal antibody-producing hybridoma according to the present invention was prepared by cell fusion of human lymphocytes stimulated in vitro and myeloma cells, and this human type monoclonal antibody is a lung adenocarcinoma cell line A549. Lung adenocarcinoma / pulmonary squamous cell carcinoma tissue sections, reacting with plasma samples from lung cancer (lung adenocarcinoma, lung squamous cell carcinoma and small cell lung cancer), lung adenocarcinoma cell line A549 Shows complement-dependent cytotoxic activity against
In addition, in vivo (in vivo) experiments, that is, transplantation of the lung cancer cell line A549 to cause tumor formation, severe combined immunodeficient mice (severe combined)
・ Immunodeficiency ・ mouse; S
In the pharmacokinetic experiment using CID), the humanized monoclonal antibody-producing hybridoma 28K29 having the property that the accumulation property of the antibody is positive and has the property of causing tumor degeneration, and the antibody class and the light chain are λ chains of IgM and the human type A monoclonal antibody was found.

The human-type monoclonal antibody of the present invention may be the human-type monoclonal antibody, the human-type monoclonal antibody fragment, or the human-type monoclonal antibody or the human-type monoclonal antibody fragment, for example, a radioisotope (RI) or a paramagnetic substance. , A photosensitizer, a toxin, or an immunocomplex bound to an anti-cancer substance, etc., can be used for a lung cancer therapeutic method / a lung cancer diagnostic method, which is a safer therapeutic agent / diagnostic agent for humans. It has been found to be able to provide.

The present invention has been completed on the basis of the above knowledge, and has been entrusted to the Institute of Biotechnology, Institute of Industrial Science with the contract number F.
Hybridoma 28K29 having the characteristics of cells deposited under ERM P-14119, humanized monoclonal antibody produced by hybridoma 28K29 having the characteristics of cells deposited under the accession number FERMP-14119 at the Institute of Biotechnology, Institute of Industrial Science and Technology , A cancer diagnostic agent containing the human type monoclonal antibody or the human type monoclonal antibody fragment as a main component, and a cancer therapeutic agent containing the human type monoclonal antibody or the human type monoclonal antibody fragment as a component.

Particularly preferably, the antigen has the following characteristics: (1) The antigen can be bound by a humanized monoclonal antibody produced by the hybridoma 28K29: (2) The antigen is small cell lung cancer cell line PC-6 It is contained in the cell extract of the strain, and exists as a major band on SDS-polyacrylamide electrophoresis under reducing and non-reducing conditions at a position of molecular weight of about 500,000 to about 800,000: (3) The antigen is present on the cell membrane of the lung adenocarcinoma cell line A549: (4) The antigen is present in the body fluid of the lung cancer patient: (5) The antigen is present in the cancer tissue of the lung cancer patient: IgM human monoclonal antibody and the Ig
This is a hybridoma 28K29 capable of producing M human type monoclonal antibody.

The present invention will be described in detail below, but the present invention is not limited thereto. The present invention resides in the production of a novel human monoclonal antibody that reacts with lung cancer, and can be obtained, for example, as follows.
That is, the lymph nodes belonging to a lung cancer patient are cut into small pieces with scissors, the insoluble portion is removed with gauze, and the cells are sensitized with cancer cells in the patient's body by low-speed centrifugation to produce antibody-producing cells (B cells).
Collect lymphocytes containing. These recovered lymphocytes are cultured in a culture medium containing a cell activating factor such as lipopolysaccharide, lectin, or killed Staphylococcus aureus at 37 ° C. for about 3 to about 8 days in the presence of 5% carbon dioxide. continue.

Then, activated lymphocytes and a fused parent strain, such as P3X63Ag8.653 (ATCC.C
RL ・ 1580) and SP2 / 0-Ag14 (ATCC ・
CRL • 1581) and other mouse myeloma cell parent lines or SHM-D33 (ATCC • CRL • 1668)
Fused cells, that is, hybridomas are prepared by cell fusion with a human-mouse heteromyeloma cell parent such as SHM-D36 or SHM-D36, for example, an electrical cell fusion method, a polyethylene glycol method, or a Seidai virus method. That is, cell fusion between the lymphocyte and the myeloma cell is performed.

For example, the lymphocytes and myeloma cells are
Mixing in a ratio of 10: 1 to 1:10, preferably 2: 1 to 1: 1 and isotonic sorbitol solution (280 mM sorbitol, 0.1 mM calcium acetate, 0.5 mM magnesium acetate, 0.1%). After washing once with bovine serum albumin), the cells mixed with a hypotonic sorbitol solution (70 mM sorbitol, 0.1 mM calcium acetate, 0.5 mM magnesium acetate, 0.1% bovine serum albumin) are resuspended and resuspended for 5 minutes. Then, by applying an electric pulse of 1.0 KV / cm for 15 microseconds, electric cell fusion (Fung, S et al., J. Immunol. Meth) was performed.
ods, 134, 35-42, 1990; Zimmer
mann, U et al., J. Immunol. Method
s, 134, 43-50, 1990).

Five minutes after the application of the electric pulse, the hypotonic sorbitol solution was removed by low-speed centrifugation, and 10 ⁵ c was added to a hypoxanthine-aminopterin-thymidine (HAT) selective medium.
re-suspend at 10 to ¹⁰ × 10 ⁵ cells / ml and dispense 0.1 ml / well into a 96-well plate. The 96-well plate is observed at 37 ° C. in a CO ₂ incubator and the culture state is appropriately observed to continue the culture for 2 to 6 weeks (for example, S. Tomiyama and Tamie Ando; Monoclonal Antibody Experiment Manual, Kodansha Scientific, 198
7 years), during this culture, for the wells in which colonies of cells could be observed with a light microscope or with the naked eye, the culture supernatants were collected, and the human-type antibody in the culture supernatants was collected. The activity assay and the reactivity assay with a cancer cell line such as lung cancer adenocarcinoma line A549 (ATCC / CCL / 185) are carried out by, for example, an enzyme immunoassay method, and further, a fluorescent antibody method and lung cancer tissue using the lung cancer cell line are performed. Positive wells are selected by the immunohistochemistry method using.

Specifically, the culture supernatant liquid is collected again from the grown wells, and the reactivity with the lung adenocarcinoma cell line A549 cells is examined (Dorren's method, Br. J. Can.
cer, 45, 491-497, 1982). That is, 10% fetal bovine serum-added RDF medium (RPMI164
0 medium: Dulbecco's MEM medium: Ham's F12 medium 2:
A medium mixed at a ratio of 1: 1; K. Aihara et al., I
n ・ Vitro ・ Cellular & Developm
Ental Biology, 24, 959-962
1988), a lung adenocarcinoma cell line A5 grown in a subconfluent state in a 96-well cell culture microplate.
49 cells in phosphate buffered saline (phosphat)
e-buffered saline; PBS buffer solution)
After washing once with PB, 0.05% glutaraldehyde added PB
100 μl of S buffer is dispensed and left at room temperature for 15 minutes.

After 15 minutes, 0.05% Tween 20 (Tw
een20) Wash with PBS buffer containing 3 times, add 200 μl of PBS buffer containing 3% bovine serum albumin, and block at room temperature for 1 hour. After blocking
Wash 5 times with PBS buffer containing 0.05% Tween 20, and dispense 50 μl each of the culture supernatant. After reacting at room temperature for 3 hours, it was washed 3 times with PBS buffer containing 0.05% Tween 20, and PB containing 0.2% bovine serum albumin
Alkaline phosphatase-labeled goat F (ab ′) ₂ anti-human immunoglobulin (Tago Co.) diluted 1000 times with S buffer is dispensed in 100 μl portions and reacted at room temperature for 1 hour.

After the reaction, the plate was washed three times, and 100 μl of an alkaline phosphatase coloring substrate solution (0.67 M p-nitrophenyl phosphate, 1 M diethanolamine, 0.5 mM magnesium chloride, pH 9.6) was dispensed at room temperature, Color was developed for 10 minutes. After coloring, 100 μl of 1N sodium hydroxide solution is added to stop the reaction, and the absorbance at 405 nm is measured by a microplate reader to examine the reactivity with the lung cancer cell line A549 cells.

Further, an immunohistochemical assay is also performed using lung cancer tissue. That is, the formalin fixation method,
AMex embedding method (Sato, Y et al., A
m. J. Pathol. , 125, 431-435, 1
986; Sato, Y et al., Am. J. Pathol. ,
140, 775-779, 1992) and fresh frozen section method, and the lung cancer specificity of the human antibody is examined by immunohistochemistry using the lung cancer tissue section.

Deparaffinization was performed with xylene and ethanol series for formalin-fixed tissue sections and with xylene and acetone series for AMeX embedding method tissue sections.
The fresh frozen tissue section and the deparaffinized AMeX tissue section are washed once with PBS buffer and then fixed with 4% paraformaldehyde (0.1 M phosphate buffer, pH 7.4) at room temperature for 20 minutes. Deparaffinized formalin-fixed slides and paraformaldehyde-fixed fresh frozen section slides or AMeX tissue section slides were washed three times with PBS buffer, and then blocked with 10% normal goat serum for 15 minutes at room temperature. (Nobuhiko Soyama et al., Nissho, 87, 2635-2641; Soya
ma, N et al., Gastroenterol. Jpn. ,
24, 581, 1989).

That is, after reacting a culture supernatant liquid sample containing a human antibody in advance with a 1/400 amount of peroxidase-labeled goat F (ab ') ₂ anti-human immunoglobulin (Cappel) at room temperature for 3 hours. To block free peroxidase-labeled goat F (ab ') ₂ anti-human immunoglobulin, one-75th amount of normal human serum (Capel) was added, and the mixture was further reacted at room temperature for 1 hour. This human antibody and peroxidase-labeled goat F (ab ')
₂ Reaction solution containing anti-human immunoglobulin conjugate
Place 2 ml on deparaffinized slide or paraformaldehyde-fixed fresh frozen section slide,
After standing still at 4 ° C for 18 hours and washing three times with a PBS buffer solution, a chromogenic substrate solution (0.02% diaminobenzidine, 0.005%
Color is developed with hydrogen peroxide solution, 50 mM Tris-HCl, pH 7.6) for 5 minutes.

After the color is developed, it is washed with water, counter-stained with 3-fold diluted Harris hematoxylene, and further thoroughly washed with water. After that, dehydration reaction was performed with 70% ethanol, 80% ethanol, 90% ethanol, 100% ethanol (2
X) and 100% xylene (twice) in this order, and mount xylene-based sample mountant mount quick (Daido Sangyo Co., Ltd.)
Enclose with. By observing the encapsulated tissue section slides with a light microscope, human type antibodies that do not react with normal sites but specifically react with lung cancer tissues are selected.

The selection by the fluorescent antibody method may be performed as follows. That is, using a cell culture flask (F75), a lung adenocarcinoma cell line A549 cells grown to a subconfluent state in Dulbecco's MEM medium containing 10% fetal bovine serum was treated with 0.02% ethylenediaminetetraacetic acid 2
Treatment with PBS buffer containing sodium (EDTA),
Adherent cells are released and cells are collected by centrifugation at 1200 RPM for 10 minutes.

150 μl of the hybridoma culture supernatant was reacted with 2 × 10 ⁵ A549 cells collected at 4 ° C. for 2 hours and then centrifuged at 1200 RPM for 10 minutes to precipitate the cells. The cells were resuspended in 100 µl of FITC-labeled goat F (ab ') ₂ anti-human immunoglobulin antibody (Capel) diluted 20 times with PBS buffer, and the cells were resuspended at 4 ° C for 1 ° C.
After reacting for a time, the cells are pelleted by centrifugation at 1200 RPM for 10 minutes. The cells may be washed with PBS buffer three times, suspended in 50 μl of PBS buffer, and observed under a fluorescence microscope.

Thus, a hybridoma producing a human monoclonal antibody that reacts with lung cancer can be easily obtained, and the hybridoma is stabilized by repeating cloning by the limiting dilution method two to three times. Can be established as a strain of hybridoma that is specifically produced. At this time, in selecting a human-type monoclonal antibody-producing hybridoma, the medium used is not particularly limited as long as the hybridoma can grow and proliferate. For example, RPMI1
640 medium, Dulbecco's MEM medium, Ham's F12 medium,
Iscove's medium, GIT medium (Nippon Pharmaceutical) or RDF medium (RPMI1640 medium: Dulbecco's MEM medium: Ham's F12 medium mixed at a ratio of 2: 1: 1; KA
ihara et al., In Vitro Cellular &
Development / Biology, 24,
959-962, 1988) and the like with 1-2 fetal bovine serum.
0% added medium or serum-free medium such as HYBR
Examples thereof include IDOMA-SFM medium (GIBCO), non-serum medium (international reagent) and SFM101 medium (Nissui Pharmaceutical).

Finally, in order to link it to an industrial antibody production method, these hybridomas are treated in a serum-free medium such as the above-mentioned Hybridoma-SFM (trade name: HYBRID).
A hybridoma strain capable of serum-free culture is prepared by acclimatization to a serum-free medium such as OMA-SFM, GIBCO) medium, non-serum medium (international reagent) and SFM101 medium (Nissui Pharmaceutical). A human type monoclonal antibody that reacts with lung cancer can be obtained by the method described above.

From the human-type monoclonal antibodies selected by the various selection methods described above, a human-type monoclonal antibody that reacts better with lung cancer is selected, and a hybridoma producing the human-type monoclonal antibody is identified as 28K29 (Biotechnology Industry, Institute of Industrial Science and Technology). Contract number FERM P-1
Deposited as No. 4119). The human monoclonal antibody produced by the hybridoma may be prepared according to the following procedure.

That is, a human type monoclonal antibody-producing hybridoma is transplanted and cultured using a conventional medium as described above. For example, the culture is carried out at 37 ° C. in a 5% CO ₂ incubator for 1 to 6 weeks. . Then, the culture is recovered, and is adsorbed and eluted using an appropriate adsorption column, for example, a hydroxyapatite column, isolated and recovered,
It may be appropriately purified by, for example, gel filtration or ion exchange chromatography.

For example, the culture supernatant liquid was used as a hydroxyapatite column (PENTAX-SH-2010C-diameter 2).
1.5 mm × 100 mm, Asahi Optical Co., Ltd.), the human-type monoclonal antibody was adsorbed on the column, and the column was thoroughly washed with 40 mM phosphate buffer (pH 6.8), then 40 mM to 400 mM phosphate buffer. (PH
The antibody portion may be eluted at about 300 mM by the linear concentration gradient elution method of 6.8), and a lyophilized product may be prepared by appropriately adding a stabilizer such as glycerin or saccharide.

Cancer diagnostic agents using the humanized monoclonal antibody according to the present invention include immunochemical cancer diagnostic agents such as RIA and EIA, in vitro diagnostic agents such as pathological diagnostic agents using pathological tissue specimens, and RI (radioactive agent). (Isotope) or paramagnetic substance is bound and labeled and administered into the patient's body, for example, γ-camera or MRI (nuclear magnetic resonance imaging)
Includes an in-vivo diagnostic agent that visualizes a tumor site with a device.

The human type monoclonal antibody may be an antibody fragment obtained by, for example, treatment with pepsin, and can be easily obtained, for example, by the following procedure. That is, purified human monoclonal antibody solution was added with 0.02M sodium acetate buffer solution (pH: 0.15M sodium chloride).
Dialysis against 4.0) for 16 hours at 4 ° C and finally 50
Prepare to a concentration of μg / ml (5 ml). Pepsin (Sigma) dissolved in the buffer solution was added to the antibody in a weight ratio of 1/25 to the antibody and reacted at 32 ° C for 60 minutes. After 60 minutes, the enzymatic reaction was 0.2M Tris-. A 1/5 volume of hydrochloric acid buffer solution (pH 8.2) is added to the reaction solution in a volume ratio to stop the reaction.

500 μl of pepsin-digested sample, Pr
otain-KW2003-GFC column (Showa Denko,
(20 mm × 300 mm), and gel filtration separation is performed by high performance liquid chromatography (HPLC). The equilibration buffer and elution buffer of the column are, for example, 0.01 M Tris-HCl buffer (pH 7.
2), using the condition of 2.5 ml / min,
The protein is detected by the absorbance at 0 nm.

A peak portion having a gel filtration molecular weight of about 130,000 was collected, and it was confirmed by SDS-polyacrylamide electrophoresis that the molecular weight was about 134,000 under non-reducing conditions. Two bands with molecular weights of about 25,000 and about 42,000 are identified. This gel filtration-like molecular weight pooled about 130,000 peaks and purified F (a
b ′) 2 may be used.

[0035] For example, as a cancer test drug by EIA,
The procedure may be as follows. Purified human monoclonal antibody or antibody fragment was added to 50 mM bicarbonate buffer (pH 8.
5) and suspended in NHS-LC-Biotin (1 mg /
ml water, Pierce Inc.) is added, and the mixture is reacted in ice for 2 hours. After the reaction, in order to remove unreacted biotin, 1000 g with Centricon-30 (Millipore, USA),
Centrifuge for 30 minutes, 150 mM sodium chloride and 0.1%
Dilute with 10 mM phosphate buffer containing sodium azide,
Perform centrifugation in the same manner. This operation is carried out three times, and finally, it is replaced with 150 mM sodium chloride and 10 mM phosphate buffer containing 0.1% sodium azide to easily obtain a biotinylated labeled antibody.

On the other hand, as an antibody for immobilization, unlabeled human monoclonal antibody or antibody fragment was added to 30 mM Tris-
Dilute to 1 μg / ml with hydrochloric acid (pH 8.8) and dispense 50 μl per well. After standing at 4 ° C. for 18 hours, the plate was washed twice with PBS buffer containing 0.02% Tween 20 and washed with PBS buffer containing 5% skim milk at room temperature for 3 times.
Block for hours. 0.02% after blocking
Wash twice with PBS buffer containing Tween 20 and, for example, a plasma sample with 0.2% bovine serum albumin and 0.02%
Dilute 10-fold with PBS buffer containing Tween 20 and dispense 50 μl each. 0.02 after 2 hours at room temperature
Wash 5 times with PBS buffer containing% Tween 20 and
PB with 5% skim milk and 0.02% Tween 20
50 μl of biotin-labeled human type monoclonal antibody solution diluted to 5 μg / ml with S buffer is dispensed.

After 2 hours at room temperature, 0.02% Tween2
Wash 5 times with PBS buffer containing 0 and PBS containing 0.5% skim milk and PBS containing 0.02% Tween 20.
50 μl of 0-fold diluted alkaline phosphatase-labeled streptavidin (Jackson) was dispensed. After 1 hour at room temperature, alkaline phosphatase chromogenic substrate solution (0.67M p-nitrophenyl phosphate, 1
M diethanolamine, 0.5 mM magnesium chloride,
100 μl of pH 9.6) is dispensed, and color is developed for 10 minutes at room temperature.

After coloring, 1N sodium hydroxide solution was added to 100
The amount of antigen in plasma of lung cancer patient may be quantitatively measured by adding μl each to stop the reaction, measuring the absorbance at 405 nm with a microplate reader. Also, for example, R
An in-vivo drug using the I-labeled human antibody may be used as follows. Examples of RI include In-111 and T
c-99m, I-131, I-123 and the like.
As an antibody labeling method by RI, diethylenetriaminepentaacetic acid (diethylenetriaminepepe) is used.
chelating method using a chelating compound such as ntaacetic acid (DTPA) or Schwarz (Schw)
arts) method (J. Nucl. Med., 31, 692).
-697, 1990), etc., but is not particularly limited, and there is no problem as long as it is easy for those skilled in the art.
The amount of the RI-labeled human monoclonal antibody administered varies depending on the age, symptoms, weight, etc. of the patient, but is about 0.1 mg to about 40 mg of the labeled antibody for mammals including humans.
Preferably, the unlabeled antibody may be administered together with 0 mg to 100 mg by intravenous injection, for example. On the 1st to about 10th day after the administration, the γ-camera may be used for imaging to depict the cancerous part.

The therapeutic drug for cancer using the human monoclonal antibody according to the present invention may be administered alone because it exhibits complement-dependent cytotoxicity (CDC) activity on human cancer cells. In addition, when a human type monoclonal antibody or an antibody fragment thereof is used, it is sufficiently effective even in the form of an immune complex to which a toxic substance such as a toxin protein, RI or an anticancer agent is bound. Examples of toxin proteins include ricin, diphtheria toxin, tumor necrosis factor, and the like.
Examples of I include I-131, Y-90, and Re-18.
6 and the like, and examples of the anticancer agent include adriamycin, vincristine, doxorubicin, and the like.

Treatment of cancer by administration of the human type monoclonal antibody alone may be performed as follows. The human monoclonal antibody of the present invention can be used alone or in combination with one or more pharmaceutically acceptable adjuvants as an antitumor agent. For example, the human monoclonal antibody may be dissolved in physiological saline or an aqueous solution of saccharides such as mannitol to be used as an appropriate cancer therapeutic agent. Alternatively, a human monoclonal antibody may be prepared as a powder injection by a conventional method.

Although the amount of the humanized monoclonal antibody to be administered varies depending on the age, symptoms, body weight, etc. of the patient, about 0.1 mg to about 20 mg of the humanized monoclonal antibody as a therapeutic agent for cancer is administered to mammals including humans. / Kg / day. Administration is performed once a day (single administration or daily administration) or intermittently 1 to 3 times a week according to a conventional method, 2.
It is performed by intravenous injection or intratumoral injection once every 3 weeks. This cancer therapeutic agent is expected to be effective in treating cancer such as lung cancer.

[0042]

EXAMPLES Next, the present invention will be explained based on examples, but the present invention is not limited thereto.

[0043]

Example 1 Human Monoclonal Antibody Reacting Specificly with Lung Cancer Tissue (1) Preparation and Cell Fusion of Human Lymphocytes Lymph nodes belonging to a lung cancer patient are aseptically removed by surgical operation, immersed in cold RPMI1640 medium, Lymph nodes are cut into small pieces with scissors, the insoluble portion is removed with double gauze, and the mixture is centrifuged at 1200 RPM for 10 minutes in a low speed centrifuge to collect the lymphocyte portion as a precipitate. The collected lymphocytes are resuspended in PBS buffer and centrifuged again at 1200 RPM for 10 minutes in a low speed centrifuge to collect the lymphocyte portion as a precipitate and wash the lymphocytes. Washed lymphocytes with 15% fetal calf serum added R
The cells were resuspended in DF medium (RPMI1640 medium: Dulbecco's MEM medium: HamF12 medium = 2: 1: 1 mixed medium) and the cell density was 4.2 × 10 ⁶ cells / ml.
As a sample, 2 ml of a lymphocyte solution to which a killed Staphylococcus aureus Kowan I strain killed bacteria (CALBIOCHEM, USA), which is a B cell activating factor, was added at a concentration of 1/10000 was dispensed into a 6-well plate.

This plate was cultured at 37 ° C. in a 5% CO 2 incubator for 5 days, and then lymphocyte cells were collected by low speed centrifugation at 1200 RPM for 10 minutes. Also, 10%
The fusion parent strain SHM-D33 (ATCC / CRL / 166), which is in a growth phase, is cultured in an RDF medium supplemented with fetal bovine serum.
Similarly, in 8), cells were collected by centrifugation. The recovered lymphocyte cells and the fused parent cell line were mixed at a ratio of 1: 1 and subjected to an electrical cell fusion method (Fung, S et al., J. Am.
Immunol. Methods, 134, 35-4
2, 1990; Zimmermann, U. et al., J. Am. Im
munol. Methods, 134, 43-50, 1
990) to perform cell fusion. That is, the mixed cells were treated with 300 L3 solution (280 mM sorbitol,
After washing once with 0.1 mM calcium acetate, 0.5 mM magnesium acetate and 1 mg / ml bovine serum albumin, the cells were collected by centrifugation, and 75 L3 solution (70 mM sorb) was collected.
itol, 0.1 mM calcium acetate, 0.5 mM magnesium acetate and 1 mg / ml bovine serum albumin) 2.
Resuspended in 5 ml.

After 5 minutes, using an electrical cell fusion device SSH-1 manufactured by Shimadzu Corporation, 1 MHz, 1.00 kV / cm,
0.83 ml / chain bar at 15 μs,
An electric pulse was applied. After the pulse, the cells were left to stand for 5 minutes, and the cell suspension was added to 30 ml of the hybridoma selection medium (100 µ
M hypoxanthine, 16 μM thymidine, 0.4 μM aminopterin, 5 μg / ml bovine insulin, 5 μg / m
l human transferrin, 5 ng / ml sodium selenite, 50 μM 2-mercaptoethanol, 20 U
/ Ml recombinant human interleukin-6, 40
% Bulb / c mouse spleen cell culture supernatant, RDF medium containing 10% fetal bovine serum), and the cell suspension was adjusted to 0.
Each 1 ml was dispensed into a 96-well cell culture microplate. The microplate was placed in a 5% carbon dioxide incubator at 37 ° C, and the culture was continued for 2 to 4 weeks. (2) Selection of lung cancer cell-reactive hybridoma A 96-well micro-plate, which had been continuously cultured for 2 to 4 weeks, was observed by an optical microscope to collect 25 μl of culture supernatant liquid in a well in which a colony was observed, and a human type The antibody productivity was assayed. That is, anti-human immunoglobulin goat antibody (Tago, USA) was diluted 1000-fold with 0.1 M bicarbonate buffer (pH 9.3), and then ELISA was performed.
Dispense 100 μl each into a 96-well plate for A (enzyme immunoassay), leave at 4 ° C. overnight (16 hours), wash 3 times with PBS buffer, and plate with PBS buffer containing 3% bovine serum albumin. Was blocked at room temperature for 1 hour.

After blocking, the plate was washed twice with PBS buffer, and 25 μl of culture supernatant and 25 μl of PBS buffer were dispensed. After reacting at 37 ° C. for 1 hour, washed with PBS buffer 3 times, and diluted with 2000% PBS buffer containing 0.2% bovine serum albumin to give peroxidase-labeled goat F
(Ab ′) ₂ anti-human immunoglobulin (Cappel, USA) was dispensed in 100 μl portions and reacted at 37 ° C. for 30 minutes. After the reaction, the plate was washed 3 times with PBS buffer, and a peroxidase coloring substrate solution (0.1 M citric acid: 0.2 MN was used.
a ₂ HPO ₄ : water = 5 ml: 5 ml: 10 ml in a buffer solution of 10 mg orthophenylenediamine / 20 ml and 10 μ
1 ・ hydrogen peroxide water / 20mL 30% solution)
The solution was dispensed in an amount of 00 μl and developed at room temperature for 10 minutes.

After color development, 100 μl of 1N hydrochloric acid was added,
The reaction was stopped, the absorbance at 490 nm was measured with a microplate reader, and assay evaluation of the antibody productivity was performed. The antibody class and light chain were determined by immunoenzymatic assay. That is, as a primary antibody, anti-human immunoglobulin goat antibody (Tago Co., USA) was used.
Dilute 1000 times with M bicarbonate buffer (pH 9.3),
100 μl on 96-well plate for LISA (enzyme immunoassay)
Dispense each and leave at 4 ° C overnight (16 hours), then PB
PB containing 3% bovine serum albumin after washing 3 times with S buffer
Plates were blocked with S buffer for 1 hour at room temperature.

After blocking, the plate was washed twice with PBS buffer, and 25 μl of culture supernatant and 25 μl of PBS buffer were dispensed. After reacting at 37 ° C. for 1 hour, the mixture was washed 3 times with PBS buffer and diluted 1000-fold with PBS buffer containing 0.2% bovine serum albumin, alkaline phosphatase-labeled goat F (ab ′) ₂ anti-human IgM (tag , USA), alkaline phosphatase labeled goat F (ab ')
₂ Anti-human IgG (Tago, USA), alkaline phosphatase labeled goat F (ab ') ₂ Anti-human IgA (Tago, USA), alkaline phosphatase labeled goat F
(Ab ') ₂ anti-human Ig λ chain (Tago, USA) or alkaline phosphatase labeled goat F (ab') ₂
100 μl of anti-human Igκ chain (Tago, USA) was dispensed and reacted at 37 ° C. for 30 minutes. After reaction, PBS
The plate was washed 3 times with a buffer solution, and 100 μl of an alkaline phosphatase chromogenic substrate solution (0.67 M p-nitrophenyl phosphate, 1 M diethanolamine, 0.5 mM magnesium chloride, pH 9.6) was dispensed at room temperature and 1
Color was developed for 0 minutes.

After coloring, 1N sodium hydroxide solution was added to 10
The reaction was stopped by adding 0 μl of each, and the absorbance at 405 nm was measured with a microplate reader to examine the antibody class and L chain. For wells positive for antibody production, the culture was expanded and a 24-well plate was used to
The culture was continued on a 5 ml scale. The culture supernatant was collected again from the grown wells, and the reactivity with the lung adenocarcinoma cell line A549 cells was examined (Dorren's method, Br.
J. Cancer, 45, 491-497, 198.
2). That is, lung adenocarcinoma cell line A549 cells grown in a sub-confluent state in a 96-well cell culture microplate in RDF medium supplemented with 10% fetal bovine serum were subjected to P
After washing once with BS buffer, 100 μl each of 0.05% glutaraldehyde-added PBS buffer was dispensed at room temperature for 1 hour.
Let stand for 5 minutes. After 15 minutes, 0.05% Tween 20
The cells were washed 3 times with the added PBS buffer, and 200 μl of PBS buffer containing 3% bovine serum albumin was added to each well, and blocking was performed at room temperature for 1 hour.

After blocking, 0.05% Tween2
Wash 5 times with PBS buffer containing 0 and add 50 μl of culture supernatant.
It was dispensed in liters. After reacting for 3 hours at room temperature, 0.05
Wash with PBS buffer containing% Tween 20 three times,
Alkaline phosphatase-labeled goat F (a) diluted 1000 times with PBS buffer containing 2% bovine serum albumin
b ') ₂ 100 anti-human immunoglobulins (Tago)
Each μl was dispensed and reacted at room temperature for 1 hour. After the reaction, the plate was washed three times and the alkaline phosphatase coloring substrate solution (0.
67 M p-nitrophenyl phosphate, 1 M diethanolamine, 0.5 mM magnesium chloride, pH
9.6) was dispensed in 100 μl aliquots to develop color at room temperature for 10 minutes.

After color development, 1N sodium hydroxide solution was added to 10
The reaction was stopped by adding 0 μl each, and the absorbance at 405 nm was measured with a microplate reader.
The reactivity with 9 cells was examined. In addition, using lung cancer tissue,
An immunohistochemical assay was also performed. That is, the lung cancer specificity of the human antibody was examined by immunohistochemistry using lung cancer tissue sections prepared by three methods including the formalin fixation method, the AMeX embedding method and the fresh frozen section method. Deparaffinization was performed with xylene and ethanol series for formalin fixed tissue sections and with xylene and acetone series for AMeX embedding method tissue sections. Fresh frozen tissue sections and deparaffinized AMeX tissue sections were washed once with PBS buffer and then washed with 4% paraformaldehyde (0.
Immobilization was carried out at room temperature for 20 minutes with 1 M phosphate buffer, pH 7.4).

Deparaffinized formalin-fixed slides and paraformaldehyde-fixed fresh frozen section slides or AMeX tissue section slides were washed 3 times with PBS buffer and then blocked with 10% normal goat serum at room temperature for 15 minutes to perform indirect. Law Modification (Nobuhiko Soyama et al., Nissho, 87, 2635-2641; Soyama, N
Gastroenterol. Jpn. , 24, 5
81, 1989).

That is, after reacting a culture supernatant liquid sample containing a human antibody in advance with 1/400 amount of peroxidase-labeled goat F (ab ′) ₂ anti-human immunoglobulin (Capel) at room temperature for 3 hours. In order to block free peroxidase-labeled goat F (ab ′) ₂ anti-human immunoglobulin, a 1/75 volume of human normal serum (Cappel) was added, and the mixture was further reacted at room temperature for 1 hour. This human antibody and peroxidase-labeled goat F (ab ')
₂ Reaction solution containing anti-human immunoglobulin conjugate
Place 2 ml on deparaffinized slide or paraformaldehyde-fixed fresh frozen section slide,
After leaving still at 4 ° C. for 18 hours and washing three times with a PBS buffer solution, a chromogenic substrate solution (0.02% diaminobenzidine, 0.005%
% Hydrogen peroxide solution, 50 mM Tris-hydrochloric acid, pH 7.
Color was developed for 5 minutes in 6).

After color development, the plate was washed with water, counter-stained with 3-fold diluted Harris Hematoxylene, and further thoroughly washed with water. Then, dehydration reaction is 70% ethanol, 80%
Ethanol, 90% ethanol, 100% ethanol (twice), and 100% xylene (twice) were performed in this order, and they were mounted with a xylene-based sample mounting agent Mount Quick (Daido Sangyo Co., Ltd.). By observing the encapsulated tissue section slides with a light microscope, human type antibodies that do not react with normal sites but specifically react with lung cancer tissues were selected. Hybridomas producing human-type antibodies selected in this manner
Subcloning was performed once to obtain a hybridoma that stably produces an antibody.

Finally, this hybridoma was acclimated to a serum-free medium HYBRIDOMA-SFM (Gibco, USA) supplemented with hypoxanthine (100 μM) and thymidine (16 μM) to produce a human type monoclonal antibody hybridoma 28K29. Got Using the culture supernatant of this hybridoma 28K29, the reactivity with the above-described lung cancer cell line A549 and the immunohistochemical study were performed again. In the examination, a human antibody (Capel) that does not react with lung cancer tissue was used in addition to the human monoclonal antibody of the present invention for comparison.

Table 1 below shows the results of immunohistological examination, and FIG. 1 shows the results of examination of reactivity with lung cancer cell line A549. FIG. 1 is a diagram showing the reactivity of human antibodies with the lung adenocarcinoma cell line A549 at the absorbance of 405 nm. N = 3
Was measured, and the average value and standard deviation were shown. hIgM
(Capel) is a humanized antibody as a negative control. Further, in Table 1, Frozen is a frozen lung cancer tissue section, AM
For eX, AMeX-fixed lung cancer tissue sections are
Represents a tissue section of a fixed lung cancer, Ad represents a tissue section of lung adenocarcinoma, and Sq represents a tissue section of lung squamous cell carcinoma, and the numerical values represent the number of positives / measured numbers.

[0057]

[Table 1]

As a result, it was revealed from FIG. 1 that the humanized monoclonal antibody produced by the hybridoma 28K29 reacts with the lung cancer cell line, and Table 1 shows that any of the three types of lung cancer tissue sections fixed by any method were used. It turned out to react well. The antibody class and light chain were λ chains in IgM as determined by immunoenzyme assay.

[0059]

Example 2 Antibody Accumulation in Tumor-Bearing Mice Using Purified Human Monoclonal Antibody (1) Purification of Human Monoclonal Antibody Hybridomas acclimated to serum-free medium HYBRIDOMA-SFM were cultured on a 2 L scale spinner flask ( 25 RPM) at 37 ° C. in a CO ₂ incubator for 10 days to obtain serum-free culture supernatant of hybridoma. As a preservative, 0.0
5% sodium azide was added to purify the human monoclonal antibody. That is, the culture supernatant was treated with a hydroxyapatite column (PENTAX-SH-2010).
C-diameter 21.5 mm × 100 mm, Asahi Optical Co., Ltd.) was directly loaded to adsorb the human monoclonal antibody to the column. After thoroughly washing with 40 mM phosphate buffer (pH 6.8), the antibody portion was eluted at about 300 mM by a linear gradient elution method from 40 mM to 400 mM phosphate buffer (pH 6.8).

Fraction reacting with lung cancer cell line A549 and having human antibody activity (around 300 mM phosphate buffer)
Were collected and purified human monoclonal antibody fraction (2 mg
16 ml of protein / ml) was obtained. It could be confirmed as a single band by SDS-electrophoresis. (2) Accumulation of human type monoclonal antibody in vivo using tumor-bearing mice SCID (6 week-old, ♀, CLEA Japan, Inc.) of severe combined immunodeficiency mouse was used for logarithmic growth of human lung cancer adenocarcinoma cell line A5.
10 ⁷ cells of 49 cells were subcutaneously injected into the dorsal side, and 8 days later, 100 μg of the humanized monoclonal antibody was intravenously injected into the tail. On day 7 after antibody administration, blood, brain, heart, lung, liver, stomach, spleen, kidney and tumor were removed.

Each of the excised organs was immersed in a PBS buffer solution to remove blood, homogenized in a PBS buffer solution containing 0.2% Tween 20, and then allowed to stand at 4 ° C. for 20 hours.
The protein was extracted. 10,000 RPM of homogenate,
After centrifugation for 30 minutes, the supernatant was used as a protein extraction sample. The blood was left at room temperature for 30 minutes and then centrifuged at 2500 RPM for 20 minutes, and the supernatant was used as a serum sample.

The amount of humanized monoclonal antibody in the serum and protein extract samples was calculated by enzyme immunoassay. That is, a 96-well microplate in which 50 μl of goat anti-human immunoglobulin (Capel) diluted 1000-fold with 30 mM Tris-hydrochloric acid buffer (pH 8.8) was dispensed into wells was allowed to stand at room temperature for 2 hours, and 0 .02% Twee
The cells were washed twice with n20-added PBS buffer, and 100 μl of PBS buffer containing 5% skim milk and 0.02% Tween 20 was dispensed to block the wells.

After 2 hours at room temperature, 0.02% Twee
The cells were washed twice with n20-added PBS buffer, and 50 μl of 2-fold diluted serum sample or protein extraction sample was dispensed. After 3 hours at room temperature, 0.02% Tween 20 added P
Wash 5 times with BS buffer, 50 μl of 0.02% Twe
Alkaline phosphatase-labeled goat F (ab ') ₂ anti-human IgM antibody diluted 1000 times with PBS containing 0.2% bovine serum albumin and en20 was dispensed.

After 2 hours at room temperature, 0.02% Tween2
The plate was washed 5 times with 0-added PBS buffer, and the alkaline phosphatase chromogenic substrate solution ((0.67M p-nitrophenyl phosphate, 1M diethanolamine, 0.5m
100 μl of M magnesium chloride, pH 9.6) was dispensed, and color was developed for 10 minutes at room temperature. After color development, 100 μl of 1N sodium hydroxide solution was added to each well to stop the reaction, and the absorbance at 405 nm was measured with a microplate reader to quantify the amount of IgM antibody.

In the examination, a human type antibody (IgM, Capel) that does not react with lung cancer tissue was used in addition to the human type monoclonal antibody of the present invention for comparison. FIG. 2 shows the results of the antibody accumulation property on the tumorous part of the tumor-bearing mouse using the human antibody. FIG. 2 shows the human-bearing antibody-bearing SCI.
FIG. 3 is a diagram showing the accumulation property to a tumor part of D mouse as a ratio (T / B) of the accumulation amount per unit weight (T) to the tumor part to the accumulation amount per unit weight (B) to blood. N = 2
Was measured, and the average value and standard deviation were shown.

As a result, in the case of the human type monoclonal antibody produced by the hybridoma 28K29, remarkable antibody accumulation in the tumor site was observed even when compared with the antibody of the negative control (IgM manufactured by Capel).
In the case of the humanized monoclonal antibody produced by K29,
Tumor regression was observed macroscopically.

[0067]

Example 3 Complement-Dependent Cytotoxicity Activity (CDC Activity) Lung cancer adenocarcinoma cells grown in a Dulbecco's MEM medium containing 10% fetal bovine serum to a subconfluent state using a cell culture flask (F75). The strain A549 cells were treated with 0.02% EDTA-added PBS buffer to release adherent cells, and the cells were collected by centrifugation at 1200 RPM for 10 minutes.

The collected cells were suspended in serum-free Dulbecco's MEM medium containing no phenol red to give 1 × 10 ⁶ cells / ml. Add 10 cell suspensions to a 96-well plate.
Aliquot 0 μl each, and add human type monoclonal antibody or human type antibody hIgM (IgM, Capel) at 10 μg / ml as a negative control, and further add 10% low toxicity rabbit complement (Sedalene, USA) as complement. And reacted at 37 ° C. in a 5% carbon dioxide incubator for 2 hours.

The number of viable cells was determined by the XTT method (P
aull, K .; D., et al. Heterocycl. Ch
em. 25, 911-914, 1988; Steve.
ns, M. G. et al., J. Immunol. Method
s, 157, 225-231, 1993). That is,
After the reaction, 50 μl each of XTT reagent (Boehringer Mannheim, Germany) was added and the reaction was continued for 4 hours. After completion of the reaction, 45 with a microplate reader
Absorbance at 0 nm is measured, and the system without antibody is 100%
As, the CDC activity of each antibody was calculated. In carrying out the examination, in addition to the human monoclonal antibody of the present invention, a human antibody (IgM, Capel) that does not react with lung cancer tissue was used for comparison. The results are shown in Fig. 3.

FIG. 3 is a humanized antibody lung adenocarcinoma cell line A54.
It is the figure which showed CDC activity with respect to 9. The CDC activity was represented by 0% in the system without antibody. The measurement was performed at N = 3, and the average value and standard deviation were shown. hIgM is a humanized antibody as a negative control. As a result, hybridoma 2
The humanized monoclonal antibody produced by 8K29 was found to have remarkable CDC activity as compared with the negative control,
It was found that the antibody alone is effective as a therapeutic agent.

[0071]

Example 4 Examination of Antigen Localization by Fluorescent Antibody Method Using a cell culture flask (F75), lung cancer adenocarcinoma cell line A549 grown in a Dulbecco's MEM medium containing 10% fetal bovine serum to a subconfluent state. The cells were treated with a PBS buffer containing 0.02% EDTA to release adherent cells, and the cells were collected by centrifugation at 1200 RPM for 10 minutes.

To 2 × 10 ⁵ A549 cells collected, 150 μl of the hybridoma culture supernatant was reacted at 4 ° C. for 2 hours and then centrifuged at 1200 RPM for 10 minutes to precipitate the cells. The cells were resuspended in 100 µl of FITC-labeled goat F (ab ') ₂ anti-human immunoglobulin antibody (Capel) diluted 20 times with PBS buffer, and the cells were resuspended at 4 ° C for 1 ° C.
After reacting for a time, the cells are pelleted by centrifugation at 1200 RPM for 10 minutes. The cells were washed 3 times with PBS buffer, suspended in 50 μl of PBS buffer, and observed under a fluorescence microscope.

As a result, the humanized monoclonal antibody produced by the hybridoma 28K29 was found to be the lung cancer adenocarcinoma cell A54.
The reaction with 9 cell membrane antigens could be observed by fluorescence microscopy.

[0074]

Example 5 Enzyme Immunoassay Using Lung Cancer Patient Plasma Samples (1) Biotinylation of Human Monoclonal Antibody Purified in Example 2 after buffer replacement with 50 mM bicarbonate buffer (pH 8.5) To 1 ml of human type monoclonal antibody-containing liquid (2 mg / ml of protein), 74 μl of N
HS-LC-Biotin (1 mg / ml water, Pierce) was added and reacted for 2 hours in ice. After the reaction, in order to remove unreacted biotin, centrifuge at 1000 g for 30 minutes with Centricon-30 (Millipore, USA),
The mixture was diluted with 10 mM phosphate buffer containing 150 mM sodium chloride and 0.1% sodium azide, and then centrifuged in the same manner. This operation was performed 3 times, and finally, it was replaced with 150 mM sodium chloride and 10 mM phosphate buffer containing 0.1% sodium azide. (2) Enzyme immunoassay using plasma sample Unlabeled human type monoclonal antibody was used as an antibody for immobilization at a concentration of 1 μg / ml with 30 mM Tris-hydrochloric acid (pH 8.8).
And diluted to 50 μl per well. 4 ℃,
After standing for 18 hours, add 0.02% Tween 20 P
Wash twice with BS buffer and add 5% skim milk to PBS
The cells were blocked with a buffer solution at room temperature for 3 hours.

After blocking, 0.02% Tween2
Washed twice with 0-added PBS buffer, 0.2% plasma sample
It was diluted 10-fold with a PBS buffer solution containing bovine serum albumin and 0.02% Tween 20 and dispensed in 50 μl aliquots. After 2 hours at room temperature, add 0.02% Tween 20 P
Wash 5 times with BS buffer, add 0.5% skim milk and 0.
5 μg / ml with PBS buffer containing 02% Tween 20
The biotin-labeled human type monoclonal antibody solution diluted to 50 μl was dispensed into each well.

After 2 hours at room temperature, 0.02% Tween2
Wash 5 times with PBS buffer containing 0 and PBS containing 0.5% skim milk and PBS containing 0.02% Tween 20.
50 μl of 0-fold diluted alkaline phosphatase-labeled streptavidin (Jackson) was dispensed. After 1 hour at room temperature, alkaline phosphatase chromogenic substrate solution (0.67M p-nitrophenyl phosphate, 1
M diethanolamine, 0.5 mM magnesium chloride,
100 μl each of pH 9.6) was dispensed, and color was developed for 10 minutes at room temperature.

After coloring, 1N sodium hydroxide solution is added to 100
The reaction was stopped by adding μl of each, and the absorbance at 405 nm was measured with a microplate reader. The results are shown in Fig. 4. FIG. 4 is a diagram showing the results of enzyme immunoassay using a lung cancer plasma sample in terms of absorbance at 405 nm.
Ad represents lung adenocarcinoma patient plasma, Sq represents lung squamous cell carcinoma patient plasma, SCLC represents lung small cell carcinoma, and No.
rmal showed normal plasma.

As a result, it was revealed that the human type monoclonal antibody produced by the hybridoma 28K29 has an antigen that reacts with the antibody in the plasma of many lung cancer patients, showing its effectiveness as an in vitro diagnostic agent for lung cancer.

[0079]

Example 6 Antibody Accumulation in Tumor-Bearing Mice Using F (ab ′) ₂ Antibody Fragment and EIA (1) Preparation of F (ab ′) ₂ Antibody Fragment by Pepsin Treatment By the method shown in Example 2. Purified human monoclonal antibody solution added with 0.15M sodium chloride 0.02M
4 ° C, 1 against sodium acetate buffer (pH 4.0)
It was dialyzed for 6 hours and finally adjusted to a concentration of 50 μg / ml (5 ml). Pepsin (Sigma) dissolved in the buffer solution was added to the antibody in a weight ratio of 1/25 to the antibody and reacted at 32 ° C for 60 minutes. After 60 minutes, the enzymatic reaction was stopped by adding 0.2 M Tris-hydrochloric acid buffer solution (pH 8.2) at a volume ratio of 1/5 to the reaction solution.

500 μl each of pepsin digested sample, Pr
otain-KW2003-GFC column (Showa Denko,
(20 mm × 300 mm), and gel filtration separation was performed. High Performance Liquid Chromatography HPLC is Shimadzu L
C-6A was used. The equilibration buffer and elution buffer of the column are 0.01 M Tris-HCl buffer (pH 7.2) containing 0.15 M sodium chloride, and the conditions are 2.5 ml / min. The protein is detected by the absorbance at 280 nm. Did.

A peak portion having a gel filtration molecular weight of about 130,000 was collected and confirmed to be about 134,000 by SDS-polyacrylamide electrophoresis under non-reducing conditions. Further, under reducing conditions, two bands having a molecular weight of about 25,000 and about 42,000 were confirmed. Therefore, about 130,000 peak portions having a gel filtration-like molecular weight were pooled and used as the purified F (ab ′) ₂ in the following experiments. (2) Antibody Accumulation in Tumor-Bearing SCID Mouse Using Purified F (ab ′) _{2 In the same} manner as in the experiment of (2) of Example 2, the human lung cancer adenocarcinoma cell line A549 was subcutaneously injected dorsally to the SCID mouse. 8 days later, the purified F (ab ′) ₂ was added to 50% of each.
Each μg was injected intravenously. On the 4th day after the antibody administration, various organs were extracted, and the human type monoclonal antibody F (ab ′) ₂ fragment accumulated in the various organs was quantified by the EIA method.

In conducting the examination, in addition to the human type monoclonal antibody of the present invention, a human type antibody which does not react with lung cancer tissue (IgM, Capel) was used for comparison. As a result, the ratio (T / B) of the accumulated amount (T) in the tumor part per unit weight to the accumulated amount (B) in the blood per unit weight is:
The case of the 28K29-F (ab ') ₂ antibody fragment is 8.5,
The value was 0.2 for the hIgM-F (ab ′) ₂ antibody fragment. Therefore, when the F (ab ′) ₂ antibody fragment was used, the accumulation property in the tumor part was remarkably observed. (3) 'as EIA immobilized antibody using _2, respectively purified F (ab purified F (ab)' with) _2, adjusted to a concentration of similarly 1 [mu] g / ml as in Example 2 (2) Then, 50 μl was dispensed per well. The same biotin-labeled antibody as in Example 2 was used.

As a result, the positive rate in the case of using the plasma of lung cancer patients was 70% in the case of 28K29-F (ab ') ₂ antibody fragment (7/10: positive sample / test sample),
In the case of the hIgM-F (ab ') ₂ antibody fragment, 0% (0 /
It was 10). Therefore, the utility as a cancer diagnostic agent using the F (ab ′) ₂ antibody fragment has been revealed.

[0084]

Example 7 Biochemical Analysis of Corresponding Antigens (1) Preparation of Cell Extract from Lung Cancer Cell Line To clarify the substance to which 28K29 antibody binds, lung cancer cell line A549 and small lung cell cell line PC6 ( Haruhiko Nakamura et al., Lung cancer, 31, 503-510, 1991) was prepared, and the molecular weight of each corresponding antigen was determined by Western blotting. A549 cells and PC6 cells were cultured in 75 cm ² cell culture flasks (4 flasks), and when they were in a subconfluent state, the cells were collected. That is, the adherent cells were washed once with PBS buffer and then with 0.02% EDTA / PBS.
Each 0 ml was dispensed into a flask and left at 37 ° C. for 10 minutes.

Thereafter, 5 ml of RDF medium supplemented with 5% fetal bovine serum was added, and the cells were collected with a cell scraper. 12
The cells were collected by low-speed centrifugation at 00 RPM for 5 minutes, and the cell extraction buffer (1% Triton X100, 5 μg / ml leupeptin, 5 μg / ml chymostatin, 5 μg / ml) was added.
PBS buffer pH 7, supplemented with 1 ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride.
4) Add 1 ml to suspend the cells, and then at 0 ° C for 45 minutes, 5
Stir every minute and let stand. After 45 minutes, 2000 rpm,
Centrifuge for 5 minutes and transfer the supernatant to a new centrifuge tube for another 10
Centrifugation was performed at 000 RPM for 30 minutes, and the centrifugation supernatant was collected.
This supernatant was used as a cell extract in Western blot experiments. (2) Western Blot 10 μl of the cell extract was added to SDS electrophoresis buffer (0.0
625M Tris-HCl pH 6.8, 10% glycerol,
Diluted 5 times with 40 μl of 2% SDS, 5% 2-mercaptoethanol [added only under reducing conditions], 0.005% bromophenol blue dye), and heat-treated at 94 ° C. for 3 minutes.

The heat-treated sample was cooled with ice and applied to the lane of SDS-polyacrylamide gel (2-15%, Daiichi Pure Chemicals Co., Ltd.) in an amount of 5 μl each. Electrophoresis was performed using electrophoresis buffer (Tris-hydroxymethyl-aminomethane 3.
0 g, glycine 14.4 g, and SDS 1 g were dissolved in distilled water to make the total amount 1 L), and about 1 at a constant current of 20 mA.
It was run for an hour. After the migration, Immobilon-P (PVDF membrane,
20% methanol-added SDS electrophoresis buffer was used in Millipore) and was blotted for 1 hour under a constant current of 100 mA using a semi-dry type blotting apparatus (Bio-Rad).

After blotting, the membrane was blocked with 5% skin milk at room temperature for 30 minutes. Then, biotinylated labeled antibody 1 μg / ml (0.5% skin milk / P
(In BS) at room temperature for 1 hour, and 0.02% Twe
The cells were washed 3 times with PBS buffer containing en20. After washing, 1 μg of alkaline phosphatase-labeled streptavidin
/ Ml (0.5% skin milk / in PBS) at room temperature,
React for 0.5 hours, add 0.02% Tween 20 P
Washed 3 times with BS buffer. After washing, BCIP / NBT
Coloring kit (Kirkegaard & Perry, N
o. 50-81-00), and after the color was developed, the reaction was stopped by washing with water.

As a result, when human type monoclonal antibody produced by hybridoma 28K29 was used, A54
9 and PC6 have a molecular weight of about 50 as a major band.
Bands at positions of 10,000 to about 800,000 (under reduced and non-reduced) were observed. Therefore, it was confirmed that these corresponding antigens were significantly expressed in the cancer cell lines.

[0089]

Industrial Applicability As described above, the humanized monoclonal antibody of the present invention has the effect that it can be used as a therapeutic agent for lung cancer, an in-vivo diagnostic agent or an in-vitro diagnostic agent.

[Brief description of drawings]

FIG. 1 is a diagram showing the reactivity of a human antibody with a lung adenocarcinoma cell line A549 by the absorbance at 405 nm.

FIG. 2 is a graph showing the accumulation of human antibodies to tumor-bearing SCID mouse tumors with respect to the accumulation amount (T) per unit weight in the tumor region relative to the accumulation amount (B) per unit weight in blood. It is a figure represented by ratio (T / B).

FIG. 3 is a diagram showing CDC activity of humanized antibodies against lung adenocarcinoma cell line A549.

FIG. 4 is a diagram showing the results of enzyme immunoassay using a lung cancer plasma sample in terms of absorbance at 405 nm.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12P 21/08 9358-4B G01N 33/53 D 33/574 D 33/577 B // (C12N 5 / 10 C12R 1:91) (C12P 21/08 C12R 1:91) (C12N 5/00 B C12R 1:91)

Claims (6)

[Claims] 1. A hybridoma 28K29 having the characteristics of the cell deposited under the deposit number FERM P-14119 at the Institute of Biotechnology, Institute of Industrial Science and Technology. 2. The hybridoma 28K29 has an antigen having the following characteristics: (1) The antigen can be bound by a humanized monoclonal antibody produced by the hybridoma 28K29: (2) the antigen is a small cell lung cancer cell line PC- It is contained in the cell extracts of 6 strains and exists as a major band on SDS-polyacrylamide electrophoresis under reducing and non-reducing, at a position of about 500,000 to about 800,000 in molecular weight: (3) Recognizing that the antigen is present on the cell membrane of lung adenocarcinoma cell line A549: (4) The antigen is present in body fluid of a lung cancer patient: (5) The antigen is present in lung cancer patient cancer tissue: The hybridoma 28K29 according to claim 1, which has the ability to produce an IgM human monoclonal antibody. 3. A humanized monoclonal antibody produced by hybridoma 28K29 having the characteristics of the cell deposited under the deposit number FERM P-14119 at the Institute of Biotechnology, Institute of Industrial Science and Technology. 4. A human monoclonal antibody having the following properties: (1) The antigen can be bound by the human monoclonal antibody produced by the hybridoma 28K29: (2) The antigen is a small cell lung cancer cell line It is contained in the cell extract of the PC-6 strain and exists as a major band on SDS-polyacrylamide electrophoresis under reducing and non-reducing conditions at a position of molecular weight of about 500,000 to about 800,000: ( 3) The antigen is present on the cell membrane of the lung adenocarcinoma cell line A549 strain: (4) The antigen is present in the body fluid of a lung cancer patient: (5) The antigen is present in the lung cancer patient cancer tissue: 4. An IgM human monoclonal antibody obtained by
The described humanized monoclonal antibody. 5. A cancer diagnostic agent comprising the human type monoclonal antibody or the human type monoclonal antibody fragment according to claim 3 as a main component. 6. A therapeutic drug for cancer comprising the human type monoclonal antibody or the human type monoclonal antibody fragment according to claim 3 as a component.